RESUMO
S4 is a paradigm of ribosomal proteins involved in multifarious activities both within and outside the ribosome. For a detailed biochemical and structural investigations of eukaryotic S4, the wheat S4 gene has been cloned and expressed in Escherichia coli, and the protein purified to a high degree of homogeneity. The 285-residue recombinant protein containing an N-terminal His(6) tag along with fourteen additional residues derived from the cloning vector is characterized by a molecular mass of 31981.24 Da. The actual sequence of 265 amino acids having a molecular mass of 29931 Da completely defines the primary structure of wheat S4. Homology modeling shows a bi-lobed protein topology arising from folding of the polypeptide into two domains, consistent with the fold topology of prokaryotic S4. The purified protein is stable and folded since it can be reversibly unfolded in guanidinium hydrochloride, and is capable of hydrolyzing cysteine protease-specific peptide-based fluorescence substrates, including Ac-DEVD-AFC (N-acetyl-Asp-Glu-Val-Asp-7-amino-4-trifluoromethylcoumarin) and Z-FR-AMC (N-CBZ-Phe-Arg-aminomethylcoumarin).
Assuntos
Escherichia coli/genética , Proteínas Ribossômicas/química , Proteínas Ribossômicas/metabolismo , Triticum/genética , Sequência de Aminoácidos , Clonagem Molecular , Cumarínicos/metabolismo , Primers do DNA , Eletroforese em Gel de Poliacrilamida , Escherichia coli/metabolismo , Guanidina , Modelos Moleculares , Dados de Sequência Molecular , Oligopeptídeos/metabolismo , Desdobramento de Proteína , Proteínas Ribossômicas/genética , Proteínas Ribossômicas/isolamento & purificação , Plântula/química , Plântula/metabolismo , Alinhamento de Sequência , Espectrometria de Fluorescência , Espectrometria de Massas em Tandem , Triticum/química , Triticum/metabolismoRESUMO
A simple and rapid reversed-phase high-performance liquid chromatographic method for the monitoring of process-related synthetic organic impurities of profenofos (PFS) is developed. Impurities are separated and determined on a reversed-phase Hypersil C(18) column using gradient elution of 50 mM ammonium formate buffer-acetonitrile as a mobile phase and detection at 230 nm at ambient temperature. The method is validated with respect to accuracy, precision, linearity, and limits of detection and quantitation. The method is found to be suitable not only for monitoring the reactions involved in the process development of PFS, but also quality assurance, as it can detect impurities at the level of 1.5 x 10(-8) g.
Assuntos
Cromatografia Líquida de Alta Pressão/métodos , Inseticidas/química , Compostos Orgânicos/análise , Organotiofosfatos/química , Reprodutibilidade dos Testes , Sensibilidade e Especificidade , Espectrofotometria UltravioletaRESUMO
The apoptotic protease activating factor (Apaf-1) is central to the regulatory mechanism by which procaspase-9 is activated in the cytochrome c-mediated pathway of apoptosis. For a detailed biochemical and structural investigation of Apaf-1 function, we have cloned and expressed in Escherichia coli inclusion bodies the WD40-deleted protein (DeltaWD40 Apaf-1) from HepG2 cell. The construct contains an N-terminal His6 tag derived from the cloning vector so that the mass of the protein and the tag together is 51,594 Da, as determined by TOF/TOF mass spectrometric analysis. An optimized refolding protocol has allowed protein recovery in highly pure form. Basic fluorescence and CD probes indicate that the refolded protein retains secondary and tertiary structures, and unfolds in the presence of higher concentration of denaturant. The equilibrium ATP binding property of the protein has been measured by changes in fluorescence emission due to the fluorescent ATP analog, mant-ATP (2'(3')-O-(N-methylanthraniloyl) adenosine 5'-triphosphate). The results demonstrate a tight Apaf-1-ATP interaction, the binding affinity being 380 nM.